Calmodulin, the primary vehicle for calcium mediated signal transduction in eukaryotes, is able to bind a large number of target proteins diverse in structure, sequence, and function. Targets are bound with a broad range of affinities, driven by varying enthalpic/entropic contributions. How a small and structurally simple protein such as calmodulin can show such complex modes of recognition is a long standing question in biochemistry. The goal of the proposed project is to understand the structural basis for calmodulin molecular recognition through crystallographic and NMR structural analysis and protein design experiments. Structure determination of CaM-peptide complexes will provide direct insights into interface properties and the nature of the physical forces driving binding and associated conformational changes. Protein design experiments to engineer CaM variants with altered binding specificities will test the validity of the model. The contributions of CaM flexibility and dynamics to recognition will be quantitatively assessed using a novel method of crystallographic analysis and compared to NMR data. Characterizing the dynamics and flexibility of CaM is essential in understanding the physical and thermodynamic bases for binding specificity. Understanding calmodulin binding specificity provides us direct insight into how calmodulin regulates the activity of an enormous number of biomedically important proteins. Designing new specificity affords the opportunity to make CaM variants that bind new cellular targets.